Polymorphism in the Apo(a) gene predict responsiveness to acetylsalicylic acid treatment

ABSTRACT

This invention relates to nucleotide polymorphisms in the human Apo(a) gene and to the use of Apo(a) nucleotide polymorphisms in identifying whether a human subject will respond or not to treatment with acetylsalicylic acid.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/477,206, filed on Apr. 3, 2017, which is a continuation of U.S.application Ser. No. 14/534,516, filed on Nov. 6, 2014, now abandoned,which is a continuation of U.S. patent application Ser. No. 13/741,750,filed Jan. 15, 2013, now abandoned, which is a continuation of U.S.patent application Ser. No. 13/090,116, filed Apr. 19, 2011, nowabandoned, which is a continuation of U.S. patent application Ser. No.12/118,060 filed May 9, 2008, now U.S. Pat. No. 7,943,317, which claimsthe benefit under 35 U.S.C. § 119(e) of U.S. provisional patentapplication No. 60/916,858, filed May 9, 2007, the disclosures of all ofwhich are incorporated herein by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant numbers HL043851 and CA 047988 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to methods for analyzing nucleotide variations inthe apolipoprotein(a) (Apo(a)) gene to evaluate a human subject'sresponsiveness to acetylsalicylic acid therapy.

BACKGROUND

Lipoprotein(a) (Lp(a)) is a plasma complex that consists of a singleapolipoprotein(a) (Apo(a)) molecule covalently linked through adisulfide bond to a single apolipoprotein B-100 molecule together withcholesterol-rich lipid (Marcovina et al. in Handbook of lipoproteintesting Rifai et al. Eds.: AACC Press, Washington, D.C., 2000; p. 819).While the biological functions of Lp(a) in normal physiology remainuncertain, high levels of Lp(a) have been associated with increasedcardiovascular risk and cardiovascular events such as myocardialinfarction and stroke, particularly when LDL-C is also elevated(Berglund et al., Arterioscler Thromb Vasc Biol 24, 2219 (2004); Hobbset al., Curr Opin Lipidol 10, 225 (1999); Danesh et al., Circulation102, 1082 (2000); Ridker et al., JAMA 297, 611 (2007); Danik et al.,JAMA 296, 1363 (2006)).

The apolipoprotein(a) locus is among the most polymorphic in the humangenome. Apo(a) genetic variation has been associated with the wide rangeof Lp(a) levels and largely accounts for the heritability of Lp(a)(Broeckel et al., Nat Genet 30, 210 (2002); Boerwinkle et al., J ClinInvest 90, 52 (1992); Mooser et al., Am J Hum Genet 61, 402 (1997);Schmidt et al., Eur J Hum Genet 14 190 (2006)). Recently, several singlenucleotide polymorphisms (SNPs) in the Apo(a) gene were identified andhave been associated with cardiovascular disorders such as myocardialinfarction and stroke and/or drug response such as response to statins(See U.S. Patent Application Publication US2005/0272054A1).

Subjects at increased risk of future cardiovascular events are oftenprescribed acetylsalicylic acid (aspirin) to reduce the risk of acardiovascular event. However, acetylsalicylic acid is not effective inall subjects and the use of acetylsalicylic acid (aspirin) as primaryprevention against cardiovascular events has been controversial,particularly in women, for whom there have been few data (Ridker et al.,N Engl J Med 352, 1293; 2005).

Thus, there is a continuing need to improve pharmaceutical agentselection design and therapy. In that regard, SNPs can be used toidentify patients most suited to treatment with particularpharmaceutical agents such as acetylsalicylic acid and/or otheranti-platelet and/or antithrombotic agents (this is often termed“pharmacogenetics”). Similarly, SNPs can be used to exclude patientsfrom certain treatments due to the patient's increased likelihood ofdeveloping toxic side effects or their likelihood of not responding tothe treatment. By doing so, such SNPs could be useful in defining thebenefit to risk ratio of a given intervention for individual subjects.Pharmacogenetics can also be used in pharmaceutical research to assistthe drug development and selection process. (Linder et al., ClinicalChemistry, 43, 254 (1997); Marshall, Nature Biotechnology, 15, 1249(1997); International Patent Application WO 97/40462, SpectraBiomedical; and Schafer et al., Nature Biotechnology, 16, 3(1998)).

SUMMARY OF THE INVENTION

The present invention relates, in part, to methods of evaluating a humansubject's response to acetylsalicylic acid treatment to reduce the riskof a future cardiovascular event. The invention is based, in part, onthe finding that novel nucleotide polymorphisms in the Apo(a) gene allowan inference to be drawn as to whether a human subject will respond ornot to treatment with acetylsalicylic acid, a particular dose ofacetylsalicylic acid, or to another anti-platelet or antithromboticagent. The invention permits identifying subjects who will respond andsubjects who will not respond to treatment with acetylsalicylic acidprior to initiation of therapy. The invention also permits selectingamong anti-platelet and antithrombotic agents the agents most likely tooffer the highest benefit of lowering risk of a cardiovascular event fora particular subject. The polymorphisms disclosed are also useful astargets for the design of diagnostic reagents and the development oftherapeutic agents for use in the diagnosis and treatment ofcardiovascular events and related pathologies.

According to one aspect of the invention, a method is provided forevaluating a human subject's responsiveness to acetylsalicylic acidtreatment to reduce the risk of a future cardiovascular event. Themethod involves determining the identity of a single nucleotidepolymorphism at position chromosome 6:160880877 (March 2006assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI) of the human subject'sapolipoprotein(a) (Apo(a)) gene. In some important embodiments, thepresence of a polymorphism characterized by cytosine or guanine at theposition chromosome 6:160880877 indicates responsiveness toacetylsalicylic acid treatment. In other important embodiments, thepresence of a polymorphism characterized by thymine or adenine at theposition chromosome 6:160880877 indicates non-responsiveness toacetylsalicylic acid treatment.

Any of a variety of detection methods may be employed, as will be wellknown to those of ordinary skill in the art. Common methods includecontacting a nucleic acid obtained from the subject with a nucleic acidprobe or sequencing a nucleic acid obtained from the subject. Examplesof such methods include but are not limited to allele-specific probehybridization, allele-specific primer extension, allele-specificamplification, 5′ nuclease digestion, molecular beacon assay,oligonucleotide ligation assay, size analysis, and single-strandedconformation polymorphism. In some important embodiments, the identityof the polymorphism is determined by sequencing a nucleic acid obtainedfrom the subject.

According to another aspect of the invention, an assay is provided. Theassay involves contacting an agent with an isolated Apo(a) proteinencoded by an Apo(a) gene having nucleotide cytosine or guanine atchromosome 6:160880877 (March 2006 assembly-NCBI build 36.1; rs3798220dbSNP @ NCBI), evaluating binding of the agent to the isolated Apo(a)protein or to Lipoprotein(a) (Lp(a)), and comparing the binding to acontrol. In some embodiments, the control involves a measurement ofbinding of a acetylsalicylic acid to the isolated Apo(a) protein or toLp(a), or to platelets or a measurement of acetylsalicylic acidinteraction with platelets.

According to another aspect of the invention, an assay is provided. Theassay involves contacting an agent with an isolated Apo(a) proteinencoded by an Apo(a) gene having nucleotide thymine or adenine atchromosome 6:160880877 (March 2006 assembly-NCBI build 36.1; rs3798220dbSNP @ NCBI), evaluating binding of the agent to the isolated Apo(a)protein, and comparing the binding to a control. In some embodiments,the control involves a measurement of binding of a acetylsalicylic acidto the isolated Apo(a) protein or to Lp(a), or to platelets or ameasurement of acetylsalicylic acid interaction with platelets.

According to another aspect of the invention, a method of treatment isprovided. The method involves selecting a human subject on the basisthat the human subject has an Apo(a) polymorphism characterized bycytosine or guanine at chromosome 6:160880877 (March 2006 assembly—NCBIbuild 36.1; rs3798220 dbSNP @ NCBI) and administering to the subjectacetylsalicylic acid for reducing the risk of a future cardiovascularevent because the subject has the polymorphism. In some embodiments, thesubject also has an elevated level of Lp(a) in the blood.

According to another aspect of the invention, a method of treatment isprovided. The method involves selecting a human subject on the basisthat the human subject has an Apo(a) polymorphism characterized bythymine or adenine at chromosome 6:160880877 (March 2006 assembly—NCBIbuild 36.1; rs3798220 dbSNP @ NCBI) and administering to the subject anantithrombotic agent other than acetylsalicylic acid for reducing therisk of a future cardiovascular event because the subject has thepolymorphism. In some embodiments, the subject also has an elevatedlevel of Lp(a) in the blood.

The antithrombotic agent may be a thienopyridine or a thienopyridinederivative. Examples of thienopyridine or thienopyridine derivativesinclude but are not limited to clopidogrel, clopidogrel bisulfate,ticlopidine, prasugrel (CS-747, or LY 640315), SR 25989, and PCR 4099.Antithrombotic agents also include but are not limited to cenoxaparinsodium, ximelagatran, abciximab, otirofiban. Examples of otherantithrombotic agents also include plasminogen activator (e.g.,Activase, Alteplase) (catalyzes the conversion of inactive plasminogento plasmin. This may occur via interactions of prekallikrein,kininogens, Factors XII, Mina, plasminogen proactivator, and tissueplasminogen activator TPA), Streptokinase, Urokinase, AnisoylatedPlasminogen-Streptokinase Activator Complex, Pro-Urokinase, (Pro-UK),rTPA (alteplase or activase; r denotes recombinant), rPro-UK,Abbokinase, Eminase, Sreptase, Anagrelide, Anagrelide Hydrochloride,Bivalirudin, Dalteparin Sodium, Danaparoid Sodium, DazoxibenHydrochloride, Efegatran Sulfate, Enoxaparin Sodium, Ifetroban,Ifetroban Sodium, Tinzaparin Sodium, retaplase, Trifenagrel, Warfarin,Dextrans, aminocaproic acid (Amicar), and tranexamic acid (Amstat),Sulfinpyrazone, Dipyridamole, Clofibrate, Pyridinol Carbamate, PGE,Glucagon, Antiserotonin drugs, Caffeine, Theophyllin Pentoxifyllin, andTiclopidine. Antithrombotic agents also include those that specificallybind to platelet receptors including but not limited to the PAR-1 andPAR-2 receptors, as well as platelet receptors for thrombin, and theplatelet ADP receptors such as P2Y₁₂.

According to another aspect of the invention, a method is provided forevaluating a human subject's responsiveness to acetylsalicylic acidtreatment to reduce the risk of a future cardiovascular event. Themethod involves detecting the presence or absence of a genetic markerlinked or in linkage disequilibrium with a single nucleotidepolymorphism (SNP) at position chromosome 6:160880877 (March 2006assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI) of the human subject'sapolipoprotein(a) (Apo(a)) gene. The genetic marker may be an allele, aSNP, a restriction fragment length polymorphism (RFLP), a randomamplified polymorphic DNA (RAPD), an amplified fragment lengthpolymorphism (AFLP), or a simple sequence repeat (SSR).

In some embodiments, the genetic marker is a SNP at position chromosome6:160849894 (NCBI build 128; rs9457931 dbSNP@NCBI). In some embodiments,the genetic marker is a SNP at position chromosome 6:160830272 (NCBIbuild 128; rs9457927 dbSNP@NCBI). In some embodiments, the linkage isbetween 16, 17, or 18 repeats of Kringle (Kr) IV type 2 domain.

In some important embodiments, the presence of a polymorphismcharacterized by cytosine or guanine at the position chromosome6:160880877 indicates responsiveness to acetylsalicylic acid. In otherimportant embodiments, the presence of a polymorphism characterized bythymine or adenine at the position chromosome 6:160880877 indicatesnon-responsiveness to acetylsalicylic acid. In some embodiments, themethod further involves determining a level of Lipoprotein(a) (Lp(a)) ina blood sample from the subject.

According to yet another aspect of the invention, a method of treatmentis provided. The method involves selecting a human subject on the basisthat the human subject has a genetic marker linked or in linkagedisequilibrium with an Apo(a) polymorphism characterized by cytosine orguanine at chromosome 6:160880877 (March 2006 assembly—NCBI build 36.1;rs3798220 dbSNP @ NCBI) and administering to the subject acetylsalicylicacid for reducing the risk of a future cardiovascular event because thesubject has the polymorphism.

The genetic marker may be an allele, a SNP, a restriction fragmentlength polymorphism (RFLP), a random amplified polymorphic DNA (RAPD),an amplified fragment length polymorphism (AFLP), or a simple sequencerepeat (SSR). In some embodiments, the human subject also has anelevated level of Lipoprotein(a) (Lp(a)) in the blood.

In some embodiments, the genetic marker is a SNP at position chromosome6:160849894 (NCBI build 128; rs9457931 dbSNP@NCBI). In some embodiments,the genetic marker is a SNP at position chromosome 6:160830272 (NCBIbuild 128; rs9457927 dbSNP@NCBI). In some embodiments, the linkage isbetween 16, 17, or 18 repeats of Kringle (Kr) IV type 2 domain.

According to yet another aspect of the invention, a method of treatmentis provided. The method involves selecting a human subject on the basisthat the human subject has a genetic marker linked or in linkagedisequilibrium with an Apo(a) polymorphism characterized by thymine oradenine at chromosome 6:160880877 (March 2006 assembly—NCBI build 36.1;rs3798220 dbSNP @ NCBI), and administering to the subject anantithrombotic agent other than acetylsalicylic acid for reducing therisk of a future cardiovascular event because the subject has thepolymorphism. In some embodiments, the human subject also has anelevated level of Lipoprotein(a) (Lp(a)) in the blood.

The genetic marker may be an allele, a SNP, a restriction fragmentlength polymorphism (RFLP), a random amplified polymorphic DNA (RAPD),an amplified fragment length polymorphism (AFLP), or a simple sequencerepeat (SSR). In some embodiments, the human subject also has anelevated level of Lipoprotein(a) (Lp(a)) in the blood.

According to still another aspect of the invention, a method is providedfor evaluating a human subject's risk of a future cardiovascular event.The method involves determining the identity of a single nucleotidepolymorphism at position chromosome 6:160880877 (March 2006assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI) of the human subject'sApo(a) gene, and determining a level of Lipoprotein(a) (Lp(a)) in ablood sample from the human subject. In some embodiments, the presenceof a polymorphism characterized by cytosine or guanine at the positionchromosome 6:160880877 (March 2006 assembly—NCBI build 36.1; rs3798220dbSNP @ NCBI) of the human subject's Apo(a) gene and the presence of anelevated level of Lp(a) in the blood sample from the subject indicatesthat the subject is at an elevated risk of a future cardiovascularevent.

The following embodiments apply equally to the various aspects of theinvention set forth herein unless indicated otherwise.

The cardiovascular event may be myocardial infarction, stroke, acutecoronary syndrome, myocardial ischemia, chronic stable angina pectoris,unstable angina pectoris, cardiovascular death, coronary re-stenosis,coronary stent re-stenosis, coronary stent re-thrombosis,revascularization, angioplasty, transient ischemic attack, pulmonaryembolism, vascular occlusion, or venous thrombosis.

In some embodiments, the method further involves determining a level ofLp(a) in a blood sample from the subject. In some embodiments, thesubject has an elevated level of Lp(a) in the blood. The level of Lp(a)may be about 10 mg/dl or higher, about 15 mg/dl or higher, about 20mg/dl or higher, about 25 mg/dl or higher, about 30 mg/dl or higher,about 35 mg/dl or higher, about 40 mg/dl or higher, about 45 mg/dl orhigher, about 50 mg/dl or higher in the blood sample from the subject.Any of a variety of methods may be employed for determining the level ofLp(a). Such methods are known to those of ordinary skill in the art. Anexample of a method for determining the level of Lp(a) is described byDanik et al., JAMA 296, 1363 (2006).

These and other aspects of the invention are described in greater detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D are sets of graphs showing the relation between the Lp(a)and rs3798220 genotype. FIG. 1A) Lp(a) levels among Caucasian femalestudy participants from the WHS (median=10.3 mg/dL), FIG. 1B) Lp(a)levels as in FIG. 1A) among the three genotypes of rs3798220. Theinter-quartile ranges (IQR) and medians for the three genotypes areindicated by the boxes and their mid-lines. The whiskers span the rangeof Lp(a) values as far as 1.5 times the IQR from the median, and extremeLp(a) values beyond the whiskers are indicated by circles. FIG. 1C)Lp(a) levels for Caucasian women with heterozygous genotype (median=79.3mg/dL). Models for the distribution of Lp(a) in subpopulations with lowLp(a) and high Lp(a) are indicated by the fitted log-normal and normaldistributions, respectively. FIG. 1D) Lp(a) distribution among Caucasianmales from the PHS with heterozygous genotype for rs3798220 (median=66.9mg/dL, measured with a different assay than for the samples from theWHS, see methods).

FIGS. 2A to 2D are sets of graphs showing the attenuation of risk fromrs3798220 or elevated Lp(a) by aspirin therapy. Kaplan-Meier estimatesof the cumulative fraction of Caucasian WHS participants with incidentvascular disease FIG. 2A) stratified by rs3798220 genotype and aspirinor placebo assignment during the WHS trial for the composite endpoint ofmajor vascular events, FIG. 2B) as in FIG. 2A) but for the endpoint ofmyocardial infarction, FIG. 2C) as in FIG. 2A) but for the endpoint ofischemic stroke, and FIG. 2D) among non-carriers of the minor allele ofrs3798220 (TT genotype) stratified by Lp(a) levels above or below the90^(th) percentile (65.1 mg/dL) and aspirin or placebo assignment forthe composite endpoint of major vascular events.

FIGS. 3A to 3B are sets of histograms showing distribution of KrIV2rnumber among non-Hispanic whites. FIG. 3A) top histogram shows N Allelesvs. N Kr. IV, TYPE 2 Repeats, while bottom histogram shows fr. Allelesvs. N Kr. IV Type 2 Repeats according to genotype. FIG. 3B) N Allelesvs. N Kr. IV, TYPE 2 Repeats according to genotype.

FIG. 4 is a plot showing the distribution of Lp(a) level according tonumber of KrIV2r's among non-Hispanic whites.

FIG. 5 is a plot showing the numbers of KrIV2r's (i.e. alleles) withsignificantly different levels of Lp(a) according to rs3798220 genotypeamong non-Hispanic whites.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates, in part, to SNPs in the Apo(a) gene that areassociated with a subject's responsiveness to acetylsalicylic acidtreatment to reduce the risk of a future cardiovascular event. Theinvention also relates to the use of the polymorphism in the Apo(a) genealone or in combination with a level of Lipoprotein (a) (Lp(a)) forevaluating a human subject's response to acetylsalicylic acid treatmentand to methods of treatment based thereon. The invention is alsodirected to identifying and designing novel antithrombotic agents.

As used herein, the term “evaluate” or “evaluating”, when used inreference to a subject's responsiveness to acetylsalicylic acidtreatment, means drawing a conclusion about response to acetylsalicylicacid treatment using a process of analyzing the identity of a nucleotideat position chromosome 6:160880877 (March 2006 assembly—NCBI build 36.1;rs3798220 dbSNP @ NCBI) of the human subject's Apo(a) gene in a nucleicacid sample of the subject, and comparing the occurrence of the singlenucleotide polymorphism (SNP) to known relationships of nucleotideoccurrence(s) at position chromosome 6:160880877 (March 2006assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI). The nucleotideoccurrence can be identified directly by examining nucleic acidmolecules, or indirectly by examining a polypeptide encoded by theApo(a) gene.

Responsiveness to acetylsalicylic acid treatment means that, in anotherwise statistically similar pool of subjects, a subject onacetylsalicylic acid treatment who has a polymorphism characterized bycytosine or guanine at the position chromosome 6:160880877 (March 2006assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI) of the human subject'sapolipoprotein(a) (Apo(a)) gene is less likely to have a futurecardiovascular event than a subject who is not on acetylsalicylic acidtreatment.

In the context of the flanking sequences, the allele that is associatedwith acetylsalicylic acid responsiveness at the position chromosome6:160880877 (March 2006 assembly—NCBI build 36.1; rs3798220 dbSNP @NCBI) is, according to the University of California at Santa Cruz GenomeBrowser:

(SEQ ID NO: 1) 5′-GCTCCAAGAACAGCCTAGACACTTC C ATTTCCTGAACATGAGATTCGAGGT-3′ (SEQ ID NO: 2)3′-CGAGGTTCTTGTCGGATCTGTGAAG G TAAAGGACTTGTACTCTAA GCTCCA-5′(The plus (“+”) strand is the top strand and the minus (“−”) strand isthe bottom strand)

The allele that is associated with acetylsalicylic acidnon-responsiveness at the position chromosome 6:160880877 (March 2006assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI is, according to theUniversity of California at Santa Cruz Genome Browser:

(SEQ ID NO: 3) 5′-GCTCCAAGAACAGCCTAGACACTTC T ATTTCCTGAACATGAGATTCGAGGT-3′ (SEQ ID NO: 4)3′-CGAGGTTCTTGTCGGATCTGTGAAG A TAAAGGACTTGTACTCTAA GCTCCA-5′(The plus (“+”) strand is the top strand and the minus (“−”) strand isthe bottom strand)

The Apo (a) gene SNP is described in U.S. Patent Application PublicationUS2005272054 under the designation rs3798220 hCV25930271. The Apo (a)gene SNP may also be found by a match of the primer sequenceCGAATCTCATGTTCAGGAAAATA (SEQ ID NO:5) described in U.S. PatentApplication Publication US2005/0272054A1 the entire contents of whichare incorporated by reference herein.

The term human subject includes a human who has had a cardiovascularevent, is suspected of developing a cardiovascular event, or anasymptomatic subject who may be predisposed or at risk of a futurecardiovascular event. Thus, in some embodiments, the human subjectalready has had a primary (first) cardiovascular event, such as, forexample, a myocardial infarct or has had an angioplasty. A human subjectwho has had a primary cardiovascular event is at an elevated risk of asecondary (second) cardiovascular event. In some embodiments, the humansubject has not had a primary cardiovascular event, but is at anelevated risk of having a cardiovascular event because the human subjecthas one or more risk factors to have a cardiovascular event. In someembodiments, the subject is already on treatment with a therapy forreducing the risk of a future cardiovascular event. The therapy can beany of the therapeutic agents referred to below. In still otherembodiments, the subject has had a primary cardiovascular event and hasone or more other risk factors. In some embodiments, the human subjectis on therapy (e.g. on anti-lipemic therapy such as statin therapy) toreduce the risk of a future cardiovascular event. In some embodiments,the human subject is in the midst of an acute coronary syndrome andtreatment decisions are being made to both manage the immediatecardiovascular event and to prevent event recurrences.

Examples of risk factors for a cardiovascular event include:hyperlipidemia, obesity, diabetes mellitus, hypertension,pre-hypertension, elevated level(s) of a marker of systemicinflammation, age, a family history of cardiovascular events, andcigarette smoking. The degree of risk of a cardiovascular event dependson the multitude and the severity or the magnitude of the risk factorsthat the human subject has. Risk charts and prediction algorithms areavailable for assessing the risk of cardiovascular events in a humansubject based on the presence and severity of risk factors. One suchexample is the Framingham Heart Study risk prediction score. The humansubject is at an elevated risk of having a cardiovascular event if thesubject's 10-year calculated Framingham Heart Study risk score isgreater than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, or 20%.

Another method for assessing the risk of a cardiovascular event in ahuman subject is a global risk score that incorporates a measurement ofa level of a marker of systemic inflammation, such as CRP, into theFramingham Heart Study risk prediction score. Other methods of assessingthe risk of a cardiovascular event in a human subject include coronarycalcium scanning, cardiac magnetic resonance imaging, and/or magneticresonance angiography (Ridker et al., JAMA 297, 611, 2007).

Hyperlipidemia is hypercholesterolemia and/or hypertriglyceridemia.Hypercholesterolemic human subjects and hypertriglyceridemic humansubjects are at an increased incidence of cardiovascular events. Ahypercholesterolemic human subject is one who fits the current criteriaestablished for a hypercholesterolemic human subject. Ahypertriglyceridemic human subject is one who fits the current criteriaestablished for a hypertriglyceridemic subject. A hypercholesterolemicsubject has an LDL level of >160 mg/dL, or an LDL level >130 mg/dL andat least two risk factors selected from the group consisting of: malegender, family history of premature coronary heart disease, cigarettesmoking, hypertension, low HDL (<35 mg/dL), diabetes mellitus,hyperinsulinemia, abdominal obesity, high lipoprotein, and personalhistory of a cardiovascular event. A hypertriglyceridemic human subjecthas a triglyceride (TG) level of ≥250 mg/dL.

Hypertension is defined as a systolic blood pressure >140 mm Hg, and/ora diastolic pressure >90 mm Hg or both. Pre-hypertension is defined assystolic blood pressure between 115 and 140 mm Hg, and/or a diastolicpressure between 80 and 90 mm Hg.

Obesity is a state of excess adipose tissue mass. Although not a directmeasure of adiposity, the most widely used method to gauge obesity isthe body mass index (BMI), which is equal to weight/height² (in kg/m²)(See, e.g., Harrison's Principles of Experimental Medicine, 15thEdition, McGraw-Hill, Inc., N.Y.—hereinafter “Harrison's”). Based ondata of substantial morbidity, a BMI of 30 is most commonly used as athreshold for obesity in both men and women. A BMI between 25 and 30should be viewed as medically significant and worthy of therapeuticintervention, especially in the presence of risk factors that areinfluenced by adiposity, such as hypertension and glucose intolerance.Although often viewed as equivalent to increased body weight, this neednot be the case. Lean but very muscular individuals may be overweight byarbitrary standards without having increased adiposity. Other approachesto quantifying obesity include anthropometry (skin-fold thickness),densitometry (underwater weighing), computed tomography (CT) or magneticresonance imaging (MRI), and/or electrical impedance.

Diabetes mellitus is established in a human subject with a fastingplasma glucose level of 125 mg/dL or higher.

An elevated level(s) of a marker of systemic inflammation is a levelthat is above the average for a healthy human subject population (i.e.,human subjects who have no signs and symptoms of disease). When themarker of systemic inflammation is CRP, a CRP level of ≥1 is consideredan elevated level.

Therapies for reducing the risk of a future cardiovascular event includebut are not limited to diet and/or exercise and/or therapies with:anti-lipemic agents, anti-inflammatory agents, antithrombotic agents,fibrinolytic agents, anti-platelet agents, direct thrombin inhibitors,glycoprotein IIb/IIIa receptor inhibitors, agents that bind to cellularadhesion molecules and inhibit the ability of white blood cells toattach to such molecules (e.g. anti-cellular adhesion moleculeantibodies), alpha-adrenergic blockers, beta-adrenergic blockers,cyclooxygenase-2 inhibitors, angiotensin system inhibitor,anti-arrhythmics, calcium channel blockers, diuretics, inotropic agents,vasodilators, vasopressors, thiazolidinediones, cannabinoid-1 receptorblockers and/or any combinations thereof.

Anti-lipemic agents are agents that reduce total cholesterol, reduceLDLC, reduce triglycerides, and/or increase HDLC. Anti-lipemic agentsinclude statins and non-statin anti-lipemic agents, and/or combinationsthereof. Statins are a class of medications that have been shown to beeffective in lowering human total cholesterol, LDLC and triglyceridelevels. Statins act at the step of cholesterol synthesis. By reducingthe amount of cholesterol synthesized by the cell, through inhibition ofthe HMG-CoA reductase gene, statins initiate a cycle of events thatculminates in the increase of LDLC uptake by liver cells. As LDLC uptakeis increased, total cholesterol and LDLC levels in the blood decrease.Lower blood levels of both factors are associated with lower risk ofatherosclerosis and heart disease, and the statins are widely used toreduce atherosclerotic morbidity and mortality.

Examples of statins include, but are not limited to, simvastatin(Zocor), lovastatin (Mevacor), pravastatin (Pravachol), fluvastatin(Lescol), atorvastatin (Lipitor), cerivastatin (Baycol), rosuvastatin(Crestor), pitivastatin and numerous others.

Non-statin anti-lipemic agents include but are not limited to fibricacid derivatives (fibrates), bile acid sequestrants or resins, nicotinicacid agents, cholesterol absorption inhibitors, acyl-coenzyme A:cholesterol acyl transferase (ACAT) inhibitors, cholesteryl estertransfer protein (CETP) inhibitors, LDL receptor antagonists, farnesoidX receptor (FXR) antagonists, sterol regulatory binding protein cleavageactivating protein (SCAP) activators, microsomal triglyceride transferprotein (MTP) inhibitors, squalene synthase inhibitors, and peroxisomeproliferation activated receptor (PPAR) agonists.

Examples of fibric acid derivatives include but are not limited togemfibrozil (Lopid), fenofibrate (Tricor), clofibrate (Atromid) andbczafibratc.

Examples of bile acid sequestrants or resins include but are not limitedto colesevelam (WelChol), cholestyramine (Questran or Prevalite) andcolestipol (Colestid), DMD-504, GT-102279, HBS-107 and S-8921.

Examples of nicotinic acid agents include but are not limited to niacinand probucol.

Examples of cholesterol absorption inhibitors include but are notlimited to ezetimibe (Zetia).

Examples of ACAT inhibitors include but are not limited to Avasimibe,CI-976 (Parke Davis), CP-113818 (Pfizer), PD-138142-15 (Parke Davis),F1394, and numerous others described in U.S. Pat. Nos. 6,204,278,6,165,984, 6,127,403, 6,063,806, 6,040,339, 5,880,147, 5,621,010,5,597,835, 5,576,335, 5,321,031, 5,238,935, 5,180,717, 5,149,709, and5,124,337.

Examples of CETP inhibitors include but are not limited to Torcetrapib,CP-529414, CETi-1, JTT-705, and numerous others described in U.S. Pat.Nos. 6,727,277, 6,723,753, 6,723,752, 6,710,089, 6,699,898, 6,696,472,6,696,435, 6,683,099, 6,677,382, 6,677,380, 6,677,379, 6,677,375,6,677,353, 6,677,341, 6,605,624, 6,586,448, 6,521,607, 6,482,862,6,479,552, 6,476,075, 6,476,057, 6,462,092, 6,458,852, 6,458,851,6,458,850, 6,458,849, 6,458,803, 6,455,519, 6,451,830, 6,451,823,6,448,295, 5,512,548.

One example of an FXR antagonist is Guggulsterone. One example of a SCAPactivator is GW532 (GlaxoSmithKline).

Examples of MTP inhibitors include but are not limited to Implitapideand R-103757.

Examples of squalene synthase inhibitors include but are not limited tozaragozic acids.

Examples of PPAR agonists include but are not limited to GW-409544,GW-501516, and LY-510929.

The invention involves identifying polymorphisms in the Apo(a) gene.Polymorphisms are allelic variants that occur in a population. Thepolymorphism can be a single nucleotide difference present at a locus,or can be an insertion or deletion of one or a few nucleotides at aposition of a gene. As such, a single nucleotide polymorphism (SNP) ischaracterized by the presence in a population of one or two, three orfour nucleotides (i.e., adenine, cytosine, guanine or thymine),typically less than all four nucleotides, at a particular locus in agenome such as the human genome.

Those skilled in the art will readily recognize that nucleic acidmolecules may be double-stranded molecules and that reference to aparticular site on one strand refers, as well, to the corresponding siteon a complementary strand. In defining a SNP position, SNP allele, ornucleotide sequence, reference to an adenine, a thymine (uridine), acytosine, or a guanine at a particular site on one strand of a nucleicacid molecule also defines the thymine (uridine), adenine, guanine, orcytosine (respectively) at the corresponding site on a complementarystrand of the nucleic acid molecule. Thus, reference may be made toeither strand in order to refer to a particular SNP position, SNPallele, or nucleotide sequence. Probes and primers, may be designed tohybridize to either strand and SNP genotyping methods disclosed hereinmay generally target either strand.

Determining the identity of a nucleotide in the Apo(a) gene can beperformed, for example, by incubating the nucleic acid sample with anoligonucleotide probe or primer that selectively hybridizes to or near,respectively, a nucleic acid molecule comprising the nucleotide anddetecting selective hybridization of the primer or probe. Selectivehybridization of a probe can be detected, for example, by detectablylabeling the probe, and detecting the presence of the label using a blottype analysis such as Southern blot analysis. Selective hybridization ofa primer can be detected, for example, by performing a primer extensionreaction, and detecting a primer extension reaction product comprisingthe primer. If desired, the primer extension reaction can be performedas a polymerase chain reaction. The method can include identifying oneor more nucleotides.

Many analytical procedures may be used to detect the presence or absenceof variant nucleotides at the polymorphic positions of the invention. Ingeneral, the detection of allelic variation requires a mutationdiscrimination technique, optionally an amplification reaction andoptionally a signal generation system. A number of mutation detectiontechniques, some based on the PCR may be used in combination with anumber of signal generation systems. Many current methods for thedetection of allelic variation are reviewed by Nollau et al., Clin.Chem. 43, 1114-1120, 1997; and in standard textbooks, for example“Laboratory Protocols for Mutation Detection”, Ed. by U. Landegren,Oxford University Press, 1996 and “PCR”, 2nd Edition by Newton & Graham,BIOS Scientific Publishers Limited, 1997. Apo (a) gene SNP detection andgenotyping methods and reagents are described in U.S. Patent ApplicationPublication US2005/0272054A1 the entire contents of which areincorporated by reference herein.

To determine the percent identity of two nucleotide sequences or twoamino acid sequences of two molecules that share sequence homology, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid or aminoacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In some embodiments, at least 30%,40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a referencesequence is aligned for comparison purposes. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein, amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje; G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). In some embodiments, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch algorithm (J.Mol. Biol. (48):444-453 (1970))

In some embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In other embodiments, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12, and a gap penalty of 4.

For analyzing SNPs, it may be appropriate to use oligonucleotidesspecific for alternative SNP alleles. Such oligonucleotides which detectsingle nucleotide variations in target sequences may be referred to bysuch terms as “allele-specific oligonucleotides”, “allele-specificprobes”, or “allele-specific primers”. The design and use ofallele-specific probes for analyzing polymorphisms is described in,e.g., Mutation Detection A Practical Approach, ed. Cotton et al. OxfordUniversity Press, 1998; Saiki et al., Nature 324, 163-166 (1986);Dattagupta, EP235,726; and Saiki, WO 89/11548.

While the design of each allele-specific primer or probe depends onvariables such as the precise composition of the nucleotide sequencesflanking a SNP position in a target nucleic acid molecule, and thelength of the primer or probe, another factor in the use of primers andprobes is the stringency of the condition under which the hybridizationbetween the probe or primer and the target sequence is performed. Higherstringency conditions utilize buffers with lower ionic strength and/or ahigher reaction temperature, and tend to require a more perfect matchbetween probe/primer and a target sequence in order to form a stableduplex. If the stringency is too high, however, hybridization may notoccur at all. In contrast, lower stringency conditions utilize bufferswith higher ionic strength and/or a lower reaction temperature, andpermit the formation of stable duplexes with more mismatched basesbetween a probe/primer and a target sequence. By way of example and notlimitation, exemplary conditions for high stringency hybridizationconditions using an allele-specific probe rate as follows:Prehybridization with a solution containing 5× standard salinephosphate-EDTA (SSPE), 0.5% NaDodSO₄ (SDS) at 55° C., and incubatingprobe with target nucleic acid molecules in the same solution at thesame temperature, followed by washing with a solution containing 2×SSPE,and 0.1% SDS at 55° C. or room temperature.

Moderate stringency hybridization conditions may be used forallele-specific primer extension reactions with a solution containing,e.g., about 50 mM KCl at about 46° C. Alternatively, the reaction may becarried out at an elevated temperature such as 60° C. In anotherembodiment, a moderately stringent hybridization condition suitable foroligonucleotide ligation assay (OLA) reactions wherein two probes areligated if they are completely complementary to the target sequence mayutilize a solution of about 100 mM KCl at a temperature of 46° C.

In a hybridization-based assay, allele-specific probes can be designedthat hybridize to a segment of target DNA from one individual but do nothybridize to the corresponding segment from another individual due tothe presence of different polymorphic forms (e.g., alternative SNPalleles/nucleotides) in the respective DNA segments from the twoindividuals. Hybridization conditions should be sufficiently stringentthat there is a significant detectable difference in hybridizationintensity between alleles, and preferably an essentially binaryresponse, whereby a probe hybridizes to only one of the alleles orsignificantly more strongly to one allele. While a probe may be designedto hybridize to a target sequence that contains a SNP site such that theSNP site aligns anywhere along the sequence of the probe, the probe ispreferably designed to hybridize to a segment of the target sequencesuch that the SNP site aligns with a central position of the probe(e.g., a position within the probe that is at least three nucleotidesfrom either end of the probe). This design of probe generally achievesgood discrimination in hybridization between different allelic forms.

The nucleotide sequence (polynucleotide or oligonucleotide) can be RNAor can be DNA, which can be a gene or a portion thereof, a cDNA, asynthetic polydeoxyribonucleic acid sequence, or the like, and can besingle stranded or double stranded, as well as a DNA/RNA hybrid. Invarious embodiments, a polynucleotide, including an oligonucleotide(e.g., a probe or a primer) can contain nucleoside or nucleotideanalogs, or a backbone bond other than a phosphodiester bond. Ingeneral, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotideor oligonucleotide also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Such nucleotide analogs are well known in the artand commercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucl. Acids Res. 22: 5220-5234(1994);Jellinek et al., Biochemistry 34: 11363-11372 (1995); Pagratis et al.,Nature Biotechnol. 15:68-73 (1997), each of which is incorporated hereinby reference).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22: 977-986 (1994);Ecker and Crooke, BioTechnology 13:351360 (1995), each of which isincorporated herein by reference). The incorporation of non-naturallyoccurring nucleotide analogs or bonds linking the nucleotides or analogscan be particularly useful where the polynucleotide is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation.

A polynucleotide or oligonucleotide comprising naturally occurringnucleotides and phosphodiester bonds can be chemically synthesized orcan be produced using recombinant DNA methods, using an appropriatepolynucleotide as a template. In comparison, a polynucleotide oroligonucleotide comprising nucleotide analogs or covalent bonds otherthan phosphodiester bonds generally are chemically synthesized, althoughan enzyme such as T7 polymerase can incorporate certain types ofnucleotide analogs into a polynucleotide and, therefore, can be used toproduce such a polynucleotide recombinantly from an appropriate template(Jellinek et al., supra, 1995). Thus, the term polynucleotide as usedherein includes naturally occurring nucleic acid molecules, which can beisolated from a cell, as well as synthetic molecules, which can beprepared, for example, by methods of chemical synthesis or by enzymaticmethods such as by the polymerase chain reaction (PCR).

The test sample of nucleic acid is conveniently a sample of blood,bronchoalveolar lavage fluid, sputum, or other body fluid or tissueobtained from a subject (e.g., human). It will be appreciated that thetest sample may equally be a nucleic acid sequence corresponding to thesequence in the test sample, that is to say that all or a part of theregion in the sample nucleic acid may firstly be amplified using anyconvenient technique e.g. PCR, before analysis of allelic variation.

In various embodiments, it can be useful to detectably label apolynucleotide or oligonucleotide. Detectable labeling of apolynucleotide or oligonucleotide is well known in the art. Particularnon-limiting examples of detectable labels include chemiluminescentlabels, radiolabels, enzymes, haptens, or even unique oligonucleotidesequences.

A method of identifying a polymorphism also can be performed using aspecific binding pair member. As used herein, the term “specific bindingpair member” refers to a molecule that specifically binds or selectivelyhybridizes to another member of a specific binding pair. Specificbinding pair members include, for example, probes, primers,polynucleotides, antibodies, etc. For example, a specific binding pairmember includes a primer or a probe that selectively hybridizes to atarget polynucleotide that includes a polymorphism loci, or thathybridizes to an amplification product generated using the targetpolynucleotide as a template.

As used herein, the term “specific interaction,” or “specifically binds”or the like means that two molecules form a complex that is relativelystable under physiologic conditions. The term is used herein inreference to various interactions, including, for example, theinteraction of an antibody that binds a polynucleotide that includes apolymorphism site; or the interaction of an antibody that binds apolypeptide that includes an amino acid that is encoded by a codon thatincludes a polymorphism site.

According to methods of the invention, an antibody can selectively bindto a polypeptide that includes a particular amino acid encoded by acodon that includes a polymorphism site. Alternatively, an antibody maypreferentially bind a particular modified nucleotide that isincorporated into a polymorphism site for only certain nucleotideoccurrences at the polymorphism site, for example using a primerextension assay.

A specific interaction generally is stable under physiologicalconditions, including, for example, conditions that occur in a livingindividual such as a human or other vertebrate or invertebrate, as wellas conditions that occur in a cell culture such as used for maintainingmammalian cells or cells from another vertebrate organism or aninvertebrate organism. Methods for determining whether two moleculesinteract specifically are well known and include, for example,equilibrium dialysis, surface plasmon resonance, and the like.

Numerous methods are known in the art for determining the nucleotideoccurrence for a particular polymorphism in a sample. Such methods canutilize one or more oligonucleotide probes or primers, including, forexample, an amplification primer pair, that selectively hybridize to atarget polynucleotide, which contains one or more polymorphisms in theApo(a) gene.

An allele specific primer is used, generally together with a constantprimer, in an amplification reaction such as a PCR reaction, whichprovides the discrimination between alleles through selectiveamplification of one allele at a particular sequence position e.g. asused for ARMS™ assays. The allele specific primer is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

An allele specific primer preferably corresponds exactly with the alleleto be detected but derivatives thereof are also contemplated whereinabout 6-8 of the nucleotides at the 3′ terminus correspond with theallele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or1 of the remaining nucleotides may be varied without significantlyaffecting the properties of the primer. Primers may be manufacturedusing any convenient method of synthesis. Examples of such methods maybe found in standard textbooks, for example “Protocols forOligonucleotides and Analogues; Synthesis and Properties,” Methods inMolecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN:0-89603-247-7; 1993; 1st Edition. If required the primer(s) may belabeled to facilitate detection.

An allele-specific oligonucleotide probe may be used to detect theApo(a) gene polymorphism at the position defined herein. The design ofsuch probes will be apparent to the molecular biologist of ordinaryskill in the art. Such probes are of any convenient length such as up to50 bases, up to 40 bases, more conveniently up to 30 bases in length,such as for example 8-25 or 8-15 bases in length. In general such probeswill comprise base sequences entirely complementary to the correspondingwild type or variant locus in the gene. However, if required one or moremismatches may be introduced, provided that the discriminatory power ofthe oligonucleotide probe is not unduly affected. The probes of theinvention may carry one or more labels to facilitate detection.

Oligonucleotide probes useful in practicing a method of the inventioncan include, for example, an oligonucleotide that is complementary toand spans a portion of the target polynucleotide, including the positionof the polymorphism, wherein the presence of a specific nucleotide atthe position is detected by the presence or absence of selectivehybridization of the probe. Such a method can further include contactingthe target polynucleotide and hybridized oligonucleotide with anendonuclease, and detecting the presence or absence of a cleavageproduct of the probe, depending on whether the nucleotide occurrence atthe polymorphism site is complementary to the corresponding nucleotideof the probe.

An oligonucleotide ligation assay also can be used to identify anucleotide occurrence at a polymorphic position, wherein a pair ofprobes that selectively hybridize upstream and adjacent to anddownstream and adjacent to the site of the polymorphism, and wherein oneof the probes includes a terminal nucleotide complementary to anucleotide occurrence of the polymorphism. Where the terminal nucleotideof the probe is complementary to the nucleotide occurrence, selectivehybridization includes the terminal nucleotide such that, in thepresence of a ligase, the upstream and downstream oligonucleotides areligated. As such, the presence or absence of a ligation product isindicative of the nucleotide occurrence at the site of polymorphism.

An oligonucleotide also can be useful as a primer, for example, for aprimer extension reaction, wherein the product (or absence of a product)of the extension reaction is indicative of the nucleotide occurrence. Inaddition, a primer pair useful for amplifying a portion of the targetpolynucleotide including the SNP site can be useful, wherein theamplification product is examined to determine the nucleotide occurrenceat the polymorphism site. Particularly useful methods include those thatare readily adaptable to a high throughput format, to a multiplexformat, or to both. The primer extension or amplification product can bedetected directly or indirectly and/or can be sequenced using variousmethods known in the art. Amplification products which span apolymorphism locus can be sequenced using traditional sequencemethodologies (e.g., the “dideoxy-mediated chain termination method,”also known as the “Sanger Method” (Sanger, F., et al., J. Molec. Biol.94: 441 (1975); Prober et al. Science 238: 336-340 (1987)) and the“chemical degradation method” also known as the “Maxam-Gilbert method”(Maxam, A. M., et al., Proc. Natl. Acad. Sci. (U.S.A) 74: 560 (1977)),both references herein incorporated by reference) to determine thenucleotide occurrence at the SNP loci.

Methods that can determine the identity of a nucleotide in the Apo(a)gene include using a “microsequencing” method. Microsequencing methodsdetermine the identity of only a single nucleotide at a “predetermined”site. Such methods have particular utility in determining the presenceand identity of polymorphisms in a target polynucleotide.

Such microsequencing methods, as well as other methods for determiningthe nucleotide occurrence at a polymorphism loci are discussed inBoyce-Jacino, et al., U.S. Pat. No. 6,294,336, incorporated herein byreference.

Microsequencing methods include the Genetic Bit Analysis methoddisclosed by Goelet, P. et al. (WO 92/15712, herein incorporated byreference). Additional, primer-guided, nucleotide incorporationprocedures for assaying polymorphic sites in DNA have also beendescribed (Komher, J. S. et al, Nucl. Acids. Res. 17: 7779-7784 (1989);Sokolov, B. P., Nucl. Acids Res. 18: 3671 (1990); Syvanen, A.-C., etal., Genomics 8: 684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl.Acad. Sci. (U.S.A) 88: 1143-1147 (1991); Prezant, T. R. et al., Hum.Mutat. 1: 159-164 (1992); Ugozzoli, L. et al., GATA 9: 107-112 (1992);Nyren, P. et al., Anal. Biochem. 208: 171-175 (1993); and Wallace,WO89/10414). These methods differ from Genetic Bit. Analysis in thatthey all rely on the incorporation of labeled deoxynucleotides todiscriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidecan result in signals that are proportional to the length of the run(Syvanen, A.-C., et al. Amer. J. Hum. Genet. 52: 46-59 (1993)).

Alternative microsequencing methods have been provided by Mundy, C. R.(U.S. Pat. No. 4,656,127) and Cohen, D. et al. (French Patent 2,650,840;PCT Appln. No. WO91/02087) which discusses a solution-based method fordetermining the identity of the nucleotide of a polymorphic site. As inthe Mundy method of U.S. Pat. No. 4,656,127, a primer is employed thatis complementary to allelic sequences immediately 3′-to a polymorphicsite.

In response to the difficulties encountered in employing gelelectrophoresis to analyze sequences, alternative methods formicrosequencing have been developed. Macevicz (U.S. Pat. No. 5,002,867),for example, describes a method for determining nucleic acid sequencevia hybridization with multiple mixtures of oligonucleotide probes. Inaccordance with such method, the sequence of a target polynucleotide isdetermined by permitting the target to sequentially hybridize with setsof probes having an invariant nucleotide at one position, and a variantnucleotide at other positions. The Macevicz method determines thenucleotide sequence of the target by hybridizing the target with a setof probes, and then determining the number of sites that at least onemember of the set is capable of hybridizing to the target (i.e., thenumber of “matches”). This procedure is repeated until each member of asets of probes has been tested.

Boyce-Jacino, et al., U.S. Pat. No. 6,294,336 provides a solid phasesequencing method for determining the sequence of nucleic acid molecules(either DNA or RNA) by utilizing a primer that selectively binds apolynucleotide target at a site wherein the SNP is the most 3′nucleotide selectively bound to the target.

Accordingly, using the methods described above, the acetylsalicylic acidresponse-related haplotype allele or the nucleotide occurrence of theacetylsalicylic acid response-related SNP can be identified using anamplification reaction, a primer extension reaction, or an immunoassay.The acetylsalicylic acid response-related haplotype allele or thenucleotide occurrence of the acetylsalicylic acid response related SNPcan also be identified by contacting polynucleotides in the sample orpolynucleotides derived from the sample, with a specific binding pairmember that selectively hybridizes to a polynucleotide region comprisingthe acetylsalicylic acid response related SNP, under conditions whereinthe binding pair member specifically binds at or near theacetylsalicylic acid response-related SNP. The specific binding pairmember can be an antibody or a polynucleotide.

Antibodies that are used in the methods of the invention includeantibodies that specifically bind polynucleotides that encompasspolymorphism in the Apo(a) gene. In addition, antibodies bindpolypeptides that include an amino acid encoded by a codon that includesthe polymorphism. These antibodies bind to a polypeptide that includesan amino acid that is encoded in part by the polymorphism. Other methodsthat may be used include genotyping methods that incorporate a modifiednucleotide that is subsequently recognized by an antibody such as, forexample, Illumina's Infinium II Assay.

The antibodies specifically bind a polypeptide that includes a firstamino acid encoded by a codon that includes the polymorphism loci, butdo not bind, or bind more weakly to a polypeptide that includes a secondamino acid encoded by a codon that includes a different nucleotideoccurrence in the Apo(a) gene.

Antibodies include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F (ab′) fragments, fragments produced by aFab expression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. The immunoglobulinmolecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD,IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass of immunoglobulin molecule.

Antibodies include antibody fragments that include, but are not limitedto, Fab, Fab′ and F (ab′) 2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included are antigen-bindingfragments also comprising any combination of variable region(s) with ahinge region, CH1, CH2, and CH3 domains. The antibodies may be from anyanimal origin including birds and mammals. Preferably, the antibodiesare human, murine (e.g., mouse and rat), donkey, ship rabbit, goat,guinea pig, camel, horse, or chicken. The antibodies may bemonospecific, bispecific, trispecific or of greater multispecificity.

The antibodies may be generated by any suitable method known in the art.Polyclonal antibodies to an antigen-of-interest can be produced byvarious procedures well known in the art. For example, a polypeptide ofthe invention can be administered to various host animals including, butnot limited to, rabbits, mice, rats, etc., to induce the production ofsera containing polyclonal antibodies specific for the antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants arealso well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example; in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

After all the relevant phenotypic and genotypic information has beenobtained, statistical analyses are carried out to determine if there isany significant correlation between the presence of an allele or agenotype with the phenotypic characteristics of an individual. Allelefrequencies, Hardy-Weinberg equilibrium statistics, and linkagedisequilibrium (LD) between SNPs can be calculated. Haplotypes can thenbe calculated using, for example, an EM algorithm (Excoffier andSlatkin, Mol Biol Evol. 1995 September; 12 (S): 921-7). There are alsoother methods for inferring haplotypes that do not use the EM approachto estimate frequency. These methods are based on reconstructions ofevolutionary history, including recombination and the coalescent, andare represented in, for example, the program PHASE (Stephens, M., Smith,N., and Donnelly, P. American Journal of Human Genetics, 68, 978-989,2001). In addition to various parameters such as linkage disequilibriumcoefficients, allele frequencies, chi square statistics and otherpopulation genetic parameters such as Panmitic indices can be calculatedto control for ethnic, ancestral or other systematic variation betweenthe case and control groups.

Preferably, data inspection and cleaning are first performed beforecarrying out statistical tests for genetic association. Epidemiologicaland clinical data of the samples can be summarized by descriptivestatistics with tables and graphs. Data validation is preferablyperformed to check for data completion, inconsistent entries, andoutliers. Chi-squared tests and t-tests (Wilcoxon rank-sum tests ifdistributions are not normal) may then be used to check for significantdifferences between cases and controls for discrete and continuousvariables, respectively. To ensure genotyping quality, Hardy-Weinbergdisequilibrium tests can be performed on cases and controls separately.Significant deviation from Hardy-Weinberg equilibrium (HWE) in bothcases and controls for individual markers can be indicative ofgenotyping errors. If HWE is violated in a majority of markers, it isindicative of population substructure that should be furtherinvestigated. Moreover, Hardy-Weinberg disequilibrium in cases only canindicate genetic association of the markers with the disease (GeneticData Analysis, Weir B., Sinauer (1990)).

To test whether an allele of a single SNP is associated with the case orcontrol status of a phenotypic trait, one skilled in the art can compareallele frequencies in cases and controls. Standard chi-squared tests andFisher exact tests can be carried out on a 2×2 table (2 SNP alleles X2outcomes in the categorical trait of interest). To test whethergenotypes of a SNP are associated, chi-squared tests can be carried outon a 3×2 table (3 genotypes X2 outcomes). Score tests are also carriedout for genotypic association to contrast the three genotypicfrequencies (major homozygotes, heterozygotes and minor homozygotes) incases and controls, and to look for trends using 3 different modes ofinheritance, namely dominant (with contrast coefficients 2, −1, −1),additive (with contrast coefficients 1, 0, −1) and recessive (withcontrast coefficients 1, 1, −2). Odds ratios for minor versus majoralleles, and odds ratios for heterozygote and homozygote variants versusthe wild type genotypes are calculated with the desired confidencelimits, usually 95%.

Polymorphisms with value for distinguishing the case matrix from thecontrol, if any, can be presented in mathematical form describing anyrelationship and accompanied by association (test and effect)statistics. A statistical analysis result which shows an association ofa polymorphism marker with an acetylsalicylic acid response with atleast 80%, 85%, 90%, 95%, or 99%, most preferably 95% confidence, oralternatively a probability of insignificance less than 0.05, may beused.

In a further aspect, the diagnostic methods of the invention are used toassess the pharmacogenetics of acetylsalicylic acid treatment to reducethe risk of a future cardiovascular event. Individuals who carryparticular allelic variants of the Apo(a) gene may therefore displayaltered abilities to react to different agents or therapies such asacetylsalicylic acid treatment. This may have a direct effect on theresponse of an individual to drug therapy. The diagnostic methods of theinvention may be useful both to predict the clinical response toacetylsalicylic acid treatment and to determine therapeutic dose.

Thus, the present invention provides methods for selecting orformulating a treatment regimen (e.g., methods for determining whetheror not to administer acetylsalicylic acid to an individual methods forselecting a particular treatment regimen such as dosage and frequency ofadministration of acetylsalicylic acid, or selecting an alternativeantithrombotic treatment (i.e., other than acetylsalicylic acid) toindividuals who are predicted to be unlikely to respond toacetylsalicylic acid treatment, and methods for determining thelikelihood of experiencing toxicity or other undesirable side effectsfrom acetylsalicylic acid treatment, etc.). The present invention alsoprovides methods for selecting individuals to whom acetylsalicylic acidtreatment or other treatment will be administered based on theindividual's genotype, and methods for selecting individuals for aclinical trial of a acetylsalicylic acid treatment or other therapeuticagent based on the genotypes of the individuals (e.g., selectingindividuals to participate in the trial who are most likely to respondpositively to the acetylsalicylic acid treatment).

The SNPs of the present invention can also be used to identify noveltherapeutic targets. For example, genes containing the polymorphism ortheir products, as well as genes or their products that are directly orindirectly regulated by or interacting with these polymorphisms or theirproducts, can be targeted for the development of therapeutics. Themethods of the invention are used in the development of new drugtherapies which selectively target one or more variants of the Apo(a)gene. Identification of a link between a particular variant andpredisposition to disease development or response to drug treatment mayhave a significant impact on the design of new drugs. Drugs may bedesigned to regulate the biological activity of variants implicated indisease processes. The therapeutics may be composed of, for example,small molecules, proteins, protein fragments or peptides, antibodies,nucleic acids, or their derivatives or mimetics which modulate thefunctions or levels of the target genes or gene products.

According to another aspect of the invention, there is provided adiagnostic kit comprising an allele specific nucleotide probe of theinvention and/or an allele-specific primer of the invention. Thediagnostic kits may comprise appropriate packaging and instructions foruse in the methods of the invention. Such kits may further compriseappropriate buffer(s) and polymerase(s) such as thermostablepolymerases, for example taq polymerase.

In another aspect of the invention, the single nucleotide polymorphismsof this invention may be used as genetic markers in linkage studies.This particularly applies to the polymorphisms of relatively highfrequency. Low frequency polymorphisms may be particularly useful forhaplotyping as described below. A haplotype is a set of alleles found atlinked polymorphic sites (such as within a gene) on a single (paternalor maternal) chromosome. If recombination within the gene is random,there may be as many as 2^(n) haplotypes, where 2 is the number ofalleles at each SNP and n is the number of SNPs. One approach toidentifying mutations or polymorphisms which are correlated withclinical response is to carry out an association study using all thehaplotypes that can be identified in the population of interest. Thefrequency of each haplotype is limited by the frequency of its rarestallele, so that SNPs with low frequency alleles are particularly usefulas markers of low frequency haplotypes. As particular mutations orpolymorphisms associated with certain clinical features are likely to beof low frequency within the population, low frequency SNPs may beparticularly useful in identifying these mutations.

According to another aspect of the present invention there is provided amethod of treatment. The method of treatment comprises selecting a humansubject on the basis that the human subject has an Apo(a) polymorphismcharacterized by cytosine or guanine at chromosome 6:160880877 (March2006 assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI) and administeringto the subject a particular dose of acetylsalicylic acid or an agentother than acetylsalicylic acid (e.g. an antithrombotic agent such as athienopyridine or a thienopyridine derivative) for reducing the risk ofan adverse cardiovascular event because the subject has thepolymorphism.

Preferably determination of the status of the human subject isclinically useful. Examples of clinical usefulness include decidingwhich antithrombotic drug or drugs to administer and/or in deciding onthe effective amount of the drug or drugs. Examples of antithromboticagents are provided above.

The invention relates to methods for evaluating a human subject'sresponsiveness to acetylsalicylic treatment to reduce the risk of afuture cardiovascular event. The methods of the invention are based, inpart, determining the identity of a nucleotide at position 6:160880877(March 2006 assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI) of asubject's Apo(a) gene that, alone or in combination, allow an inferenceto be drawn as to the subject's acetylsalicylic response. Theacetylsalicylic response can be a reduction in the risk of acardiovascular disorder. As such, the compositions and methods of theinvention are useful, for example, for identifying individuals orpatients who are more or less likely to respond to acetylsalicylic acidtreatment or to treatment with an antithrombotic agent other thanacetylsalicylic acid. The compositions and methods of the invention arealso useful for predicting or determining that an individual or patientmay require an altered dose of acetylsalicylic acid or of anantithrombotic agent other than acetylsalicylic acid to reduce the riskof a cardiovascular disorder.

According to another aspect of the invention, an assay is provided. Theassay involves screening for an agent that binds preferentially toApo(a) protein (e.g., isolated Apo(a) protein) or to Lp(a) encoded by anApo(a) gene having cytosine or guanine at the position chromosome6:160880877 (March 2006 assembly—NCBI build 36.1; rs3798220 dbSNP @NCBI.

The invention also provides another assay. The assay involves screeningfor an agent that binds preferentially to Apo(a) protein (e.g., isolatedApo(a) protein) or to Lp(a) encoded by an Apo(a) gene having thymine oradenine at the position chromosome 6:160880877 (March 2006 assembly—NCBIbuild 36.1; rs3798220 dbSNP @ NCBI.

The screening assays can be used to select among antithrombotic agentsthose which preferentially bind to a polymorphism. Rational drug designmay be carried out through altering or making versions of existingantithrombotic agents and then testing the relative ability of suchversions to bind preferentially one or another polymorphism in thescreening assay of the invention. Likewise chemical libraries ofexisting or novel agents may be screened for such binding.

The skilled artisan is familiar with screening methodologies. Screeningassays include assays that measure the ability of an agent to bind aLp(a) protein. For example, screening for an agent that bindspreferentially to a polymorphism of a Lp(a) comprises contacting theagent with a polymorphism of a Lp(a), determining the binding of theagent to the polymorphism of a Lp(a), comparing the binding of the agentto the polymorphism of a Lp(a) not containing the polymorphism, whereinan increased binding of the agent to a polymorphism of a Lp(a) comparedto the binding of the agent to the polymorphism of a Lp(a) notcontaining the polymorphism indicates that the agent is anantithrombotic agent with improved efficacy in the polymorphism of the aLp(a) and wherein a decreased binding of the agent to a polymorphism ofa Lp(a) compared to the binding of the agent to the polymorphism of aLp(a) not containing the polymorphism indicates that the agent is anantithrombotic agent with reduced efficacy for the polymorphism of theLp(a). The skilled artisan is familiar with this and other screeningmethodologies including but not limited to direct binding assays,competitive binding assays, non-competitive binding assays, functionalinhibition assays, high through-put screening assays and the like.

The invention is illustrated but not limited by reference to thefollowing Examples.

EXAMPLES Example 1

Lipoprotein(a) [Lp(a)] is a plasma fraction that consists of a single(Apo(a)) molecule covalently linked through a disulfide bond to a singleapolipoprotein B-100 molecule together with cholesterol-rich lipid (1).While the biological functions of Lp(a) remain uncertain (2, 3), highlevels of Lp(a) have been associated with increased cardiovascular (CV)risk, particularly when LDL-C is also elevated (4-6). Thecholesterol-bearing aspects of Lp(a) have largely focused investigationof associated cardiovascular risk on lipid-based biology (7-11).However, apolipoprotein(a) is also highly homologous to plasminogen, andin spite of a lack of recognized proteolytic activity inapolipoprotein(a), this homology has focused alternative investigationsof cardiovascular risk on roles of Lp(a) in hemostasis, plateletfunction, and thrombosis (12-18).

The apolipoprotein(a) locus is among the most polymorphic in the humangenome. Apolipoprotcin(a) genetic variation has been associated with thewide range of Lp(a) levels and largely accounts for the heritability ofLp(a) (19-22). Most of the known variation that influences Lp(a) levelstakes the form of between 3 and 40 repeats of the Kringle (Kr) IV type 2domain located toward the amino terminus of the protein (1, 23). Thishigh degree of Kr IV type 2 polymorphism had presented a challenge formeasuring plasma Lp(a) levels until the recent development of assayreagents that recognize the single Kr V domain of apolipoprotein(a) anddo not cross-react with the Kr IV type 2 repeats (24, 25). Otherapolipoprotein(a) gene variants that have been associated with Lp(a)levels include single nucleotide polymorphisms (SNP) as well asinsertion-deletion polymorphisms, notably of a pentanucleotide sequence,TTTTA, in the promoter (26-28). In addition, polymorphism of theapolipoprotein(a) locus appears to involve copy number variation (29).

Recently, the SNP rs3798220, encoding an isoleucine to methioninesubstitution in the plasminogen protease-like domain ofapolipoprotein(a), has been suggested as a novel candidate forassociation with both Lp(a) levels and prevalent cardiovascular disease(Luke et al., submitted (30)). The genotype of this polymorphism wastherefore determined among 26,274 initially healthy women from theWomen's Health Study (WHS) who had been randomly allocated to low doseaspirin or placebo and then followed over a 10-year period for incidentcardiovascular events (31). This setting afforded the opportunity forprospective assessment not only of rs3798220 association with Lp(a)levels and vascular risk, but also of an interaction between aspirin andgenetic effects on risk. Although most of the study participants wereCaucasian, there were sufficient numbers of non-Caucasian participantsto address population-specific influences on associations betweenrs3798220 and Lp(a) levels, as have been found in previous comparisonsof other apolipoprotein(a) variants among populations with differentbiogeographic ancestry (32-36).

Among 25,038 Caucasian participants in the WHS, the minor allele ofrs3798220 in the apolipoprotein(a) gene was carried by 904 (3.6%)heterozygotes and 15 (0.06%) homozygotes for a minor allele frequency of1.9%. These genotypes represent a borderline deviation fromHardy-Weinberg equilibrium (p=0.048) due to slight deficiency ofheterozygotes and excess of homozygotes of the minor allele. Age, BMI,smoking rates, hormone replacement therapy use, and menopausal status aswell as incidence of hypertension and diabetes were not different amongthe three genotypic classes (Table 1). However, median Lp(a) levels inheterozygous (79.3 mg/dL) and homozygous carriers of the minor allele(153.9 mg/dL) were respectively eight and 15 times higher than the Lp(a)levels in non-carriers (10.0 mg/dL, p<<0.001) (Table 1, FIG. 1A, FIG.1B). This effect appeared to underlie modest but significant elevationsof the related plasma fractions total cholesterol, LDL-C, andapolipoprotein B as judged by the elimination of significance afteradjustment for Lp(a) levels. Other plasma biomarkers, including markersof inflammation and other lipid fractions, were not associated withrs3798220 genotype. Essentially identical results were found in analysescombining the heterozygotes with the homozygous carriers of the minorallele in comparisons of clinical characteristics with non-carriers(data not shown).

The distribution of Lp(a) levels among Caucasian heterozygotes wasentirely different from that among non-carriers and had a fully bimodalpattern that allowed stratification of this subpopulation into one groupwith Lp(a) greater than 27.9 mg/dL (median 92.4 mg/dL, N=627) and asecond group with Lp(a) less than 27.9 mg/dL (median 5.0 mg/dL, N=260)(FIGS. 1A-1C). All homozygous carriers of the minor allele had Lp(a)levels greater than 27.9 mg/dL, but there were too few individuals inthis group to evaluate the shape of the Lp(a) distribution (FIG. 1B).The bimodal distribution of Lp(a) among rs3798220 heterozygotes wasconfirmed in a second unrelated population of 1058 Caucasian men fromthe Physicians' Health Study (PHS) where the minor allele frequency ofrs3798220 was 1.8%, similar to its frequency in the Caucasian WHS women(FIG. 1D).

The bimodal nature of Lp(a) levels among Caucasian heterozygotes couldnot be explained by standard clinical characteristics. For example,there were no significant differences in the comparison of Caucasianheterozygous women with low or high Lp(a) in terms of age, BMI, smoking,diabetes, menopausal status, hypertension, family history of heartdisease, or use of hormone replacement therapy. Similarly, HDL-C,apolipoprotein A1, triglycerides, C-reactive protein, soluble ICAM-I,fibrinogen, creatinine, homocysteine, and HbA1c were all comparable inthe two subpopulations of the heterozygotes, and similar to the levelamong homozygotes for the major allele. Moreover, gross geographicassignment across the US did not suggest a regional basis for thebimodal Lp(a) levels. LDL-C, total cholesterol, and apolipoprotein Blevels were significantly elevated in heterozygotes with high Lp(a)compared to heterozygotes with low Lp(a) (medians: LDL-C, 130.5 mg/dL v.122.5 mg/dL; total cholesterol, 219.0 mg/dL v. 210.4 mg/dL;apolipoprotein B, 109.9 mg/dL v. 103.1 mg/dL; all p<0.01), but thedifferences were again not significant after adjustment by Lp(a). Thus,the higher levels of these lipids observed overall among heterozygotescan be explained by the increase in Lp(a). Bimodal distributions werenot observed for any of these lipids among the Caucasian heterozygousWHS participants.

The single copy of the minor allele of rs3798220 in heterozygotesconferred an approximate 50% increase in the risk of a future vascularevent among the 24,320 Caucasian study participants who collectivelyexperienced 825 cases of a first-ever event of the total CVD compositeendpoint during the 10-year follow-up period (Table 2; age adjusted HR1.50, 95% CI:1.09-2.05, p=0.012). None of the events occurred among the15 homozygous carriers of the minor allele consistent with the lowincidence rate overall, and therefore these few individuals wereexcluded from the analysis of the risk of future incident disease. Themagnitude of the effect of rs3798220 was similar for the specificendpoints of MI, ischemic stroke, and revascularization. Thus, for themore clinically relevant endpoint of major vascular events (non-fatalMI, non-fatal ischemic stroke, and cardiovascular death), theage-adjusted hazard ratio was 1.58 (95% CI: 1.07-2.33, p=0.021). Theseeffects remained statistically significant after adjustment fortraditional cardiovascular risk factors so that the fully adjustedhazard ratio for total vascular events was 1.50 (1.07-2.10, p=0.009),and the fully adjusted hazard ratio for major vascular events was 1.54(CI:1.01-2.35, p=0.043). Almost all of the increased risk amongheterozygotes was limited to those with elevated Lp(a) levels (Table 2).For example, for major cardiovascular events, the age-adjusted hazardratio for the heterozygotes with high Lp(a) [1.78 (CI:1.26-2.51,p=0.001)] was greater and more significant than for all heterozygotes,while the hazard ratio for heterozygotes with low Lp(a) [0.84(CI:0.84-1.77, p=0.65)] was less than and not statistically differentfrom the risk among noncarriers (Table 2).

The increased risk of incident cardiovascular events among Caucasianheterozygotes of rs3798220 was largely negated by allocation to low doseaspirin treatment (FIGS. 2A-2D). Specifically, among heterozygotes,random assignment to aspirin was associated with a 59% reduction inrelative risk of major vascular events (95% CI:0.81-0.10, p=0.025),whereas among non-carriers random allocation to aspirin had nosignificant effect (relative risk reduction 9%, 95% CI: 0.023 to −0.09,p=0.31) (FIG. 2A). Formally, this interaction of aspirin and genotypewas significant (p=0.05, age-adjusted model). The differential effect ofaspirin among genotype subgroups was also observed for the endpoints ofMI and ischemic stroke (clinical events associated with acute plaquerupture and occlusive thrombosis). Thus, the net genetic effect forheterozygotes in the absence of aspirin was an approximate doubling ofoverall risk for major incident vascular events combined (age-adjustedHR 2.19, 95% CI: 1.39-3.46, p=0.0007), and separately for myocardialinfarction (age-adjusted HR 2.43, 95% CI: 1.23-4.80, p=0.01) andischemic stroke (age-adjusted HR 2.41, 95% CI: 1.26-4.59, p=0.008)(FIGS. 2A-2C). However, the effect of aspirin was attenuated for theendpoint of coronary revascularization (clinical events associated withunderlying disease progression without acute occlusion; relative riskreduction 36%, 95% CI: 0.89 to −0.44, p=0.28).

The interaction of aspirin with rs3798220 genotype appeared to berelated in part to a similar interaction of aspirin with elevated Lp(a)levels in general. Among Caucasian non-carriers of the minor allele ofrs3798220, aspirin reduced the relative risk of vascular events amongthose with Lp(a) levels above the 90th percentile (65.1 mg/dL) whereasminimal effect was observed among those with Lp(a) levels below the 90thpercentile (FIG. 2D). For the revascularization endpoint, the aspirineffect among Caucasians with Lp(a) levels above the 90th percentile(relative risk reduction 39%, 95% CI: 0.64-0.05, p=0.03) was comparablein magnitude to the aspirin effect among Caucasian heterozygotes.

The prevalence of the minor allele of rs3798220 markedly differedbetween Caucasians (MAF=1.9%) and non-Caucasians in the WHS.Specifically, variation at rs3798220 was almost absent amongself-identified African-Americans (MAF=0.5%). In contrast, the minorallele was more prevalent than among Caucasians for Asian-Americans(MAF=7.6%), Hispanics (MAF=15.1%), and Native Americans (MAF=9.1%)(Table 3). The differences in allele frequency compared with Caucasianswere all significant, as were differences between African-Americans andHispanics, Asian-Americans, or Native Americans and between Hispanicsand Asians. The HapMap genotyping project detected the minor allele inAsians (CHB+JPT, MAF=5.6%) but not in Caucasians (CEU) or Africans (YRI)(37). The frequency among HapMap Asians was not significantly differentfrom the frequency in the WHS Asian-American subpopulation (p=0.59).Among Caucasians, regional differences in allele frequency potentiallyrelated to varying degrees of admixture could not be detected by grossgeographical classification across the US. In contrast to the finding inCaucasians, Lp(a) levels were neither elevated nor bimodal in theheterozygotes of the WHS Asian-American or Hispanic subpopulations, bothof which had sufficient numbers of participants to evaluate the Lp(a)distribution. Similarly, there did not appear to be an associationbetween rs3798220 and incident CVD in these populations, although powerwas limiting.

In this large-scale study of initially healthy women from the US,Caucasian carriers of the minor allele of the rs3798220 polymorphism inthe apolipoprotein(a) gene (30) had greatly elevated levels of Lp(a) aswell as a doubling of the risk of major vascular events. However, amongheterozygotes randomly allocated to low-dose aspirin, this increasedrisk of future vascular events was effectively abolished (relative riskreduction with aspirin 59%, p=0.025). Among non-carriers, by contrast,there was minimal evidence of a benefit from aspirin therapy (relativerisk reduction with aspirin 9%, p=0.31). In the context of theantithrombotic activity of aspirin, this pharmacogenetic result isconsistent with a direct role of Lp(a) in thrombosis, perhaps throughthe plasminogen protease-like domain or lysine binding functions of theKr domains as has been suggested previously (13, 38). The finding thatthe effects of aspirin on the association of rs3798220 with myocardialinfarction and stroke are greater than its effects on coronaryrevascularization raises the intriguing possibility that an interplaybetween aspirin and Lp(a) derived from a particular apoliprotein(a)allele may be more relevant for acute plaque rupture and vesselocclusion than disease progression. Whether or not the rs3798220pharmacogenetic effect also involves possible allele-dependent reductionof apolipoprotein(a) expression by aspirin remains unknown (39, 40).Still, these data provide a direct genetic method for definingsubpopulations with greater or lesser benefit from aspirin therapy, anissue pertinent to the controversial choice of aspirin or aspirinalternatives such as thienopyridines in the prevention and treatment ofvascular disease (31, 41, 42).

The molecular basis of the complex relationship between thenon-synonymous substitution encoded by rs3798220 and Lp(a) levels andcardiovascular risk will require further elucidation. In particular, thegenetic effects on Lp(a) levels and distribution make it difficult todistinguish effects on risk and aspirin response solely related toincreased Lp(a) levels from effects related to intrinsic biologicalactivity imparted by the amino acid substitution, which may alsocontribute directly to elevated Lp(a). Similarly, relationships betweenrs3798220 and haplotypes at the apolipoprotein(a) locus are unclear, butmay involve linkage to a high expressing allele of the apolipoprotein(a)locus in Caucasians, for example with an unusual number of type 2Kringle IV domains (43, 44). This hypothesis might furthermore explainthe bimodal pattern of the Lp(a) distribution, especially if rs3798220is linked to two or more haplotypes directing different levels of Lp(a).

The allele frequency analysis is consistent with at least one origin ofrs3798220 in Asia, but does not address the presence of this variant inCaucasians. Given the moderate frequency of rs3798220 in the Caucasiansand the lack of apparent regional bias in its distribution across theUS, admixture with Asian-American populations seems an unlikelyexplanation. However, while most variation private to non-Africanpopulations may have arisen through stochastic processes rather thanpositive selection (45), the association of rs3798220 with Lp(a) levelscombined with its significantly different frequency in the Caucasiansand other non-African populations raise the possibility that bothselection and drift may be responsible for its biogeographicdistribution. Both of these processes are consistent with the high levelof population-specific genetic variation and expression at theapoliprotein(a) locus as well as speculation about Lp(a) function (3).

Aside from providing clues about the biological properties of Lp(a), theassociations of rs3798220 with Lp(a) levels, cardiovascular risk, andthe effect of aspirin treatment illustrate the potential of geneticapproaches in understanding common complex disease. In this regard, theresults offer not only a pharmacogenetic strategy for managing aspecific type of CVD risk due to genetic variation but also the broaderhope of administering healthcare with the highest possible precisionthrough gene-based personalized medicine.

REFERENCES

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Study Populations: The primary study population derived from the Women'sHealth Study (WHS), a randomized trial of aspirin (100 mg orally onalternate days) and placebo in the primary prevention of cardiovasculardisease conducted among initially healthy women aged at least 45 atenrollment who were followed over a 10 year period (1, 2). Atenrollment, participants provided baseline clinical and demographicinformation. 28,345 WHS participants provided blood for plasma andgenetic analysis. Among these samples, baseline Lp(a) levels weremeasured with a turbidimetric assay that is not affected by the numberof Kr IV type-2 repeats on the Hitachi 917 analyzer (Roche Diagnostics,Indianapolis, Ind.) (3). Baseline measurement of other lipid fractions,inflammation biomarkers, and other plasma components has been describedpreviously (4). Total incident cardiovascular events adjudicated duringthe follow-up period included those typically associated with plaquerupture and vessel occlusion such as myocardial infarction (MI),ischemic stroke, and cardiovascular death, as well as those typicallyassociated with progression of underlying atherosclerotic diseasedefined as revascularization by percutaneous transluminal coronaryangioplasty (PTCA) or coronary artery bypass graft (CABG). Majorincident cardiovascular events were defined as myocardial infarction,ischemic stroke, or cardiovascular death. In addition, the associationbetween rs3798220 and plasma Lp(a) levels was evaluated in anindependent group of 1058 Caucasian men who had participated in thePhysicians Health Study (PHS) (5). Among these PHS participants, Lp(a)levels were determined with a standard clinical ELISA-based method (6).

Genotyping: Genotypes for rs3798220 in the WHS participants weredetermined by an oligonucleotide ligation procedure that combined PCRamplification of target sequences from 3 ng of genomic DNA withsubsequent allele-specific oligonucleotide ligation (7). The ligationproducts of the two alleles were then separated by hybridization toproduct specific oligonucleotides, each coupled to spectrally distinctLuminex®100™ xMAP microspheres (Luminex, Austin, Tex.). The capturedproducts were fluorescently labeled with streptavidin R-phycoerythrin(Prozyme, San Leandro, Calif.), sorted on the basis of microspherespectrum, and detected by a Luminex®100™ instrument (8). Genotypes atrs3798220 in the PHS were determined with a fluorescence-based, allelespecific, real-time DNA amplification method as described (ABI, FosterCity) (9). For the WHS and PHS populations respectively, the fractionsof samples with successful genotype determination were 96.3% and 97.8%.Of the 27,289 women with DNA samples for genotyping, full ascertainmentfor rs3798220 genotype, Lp(a) levels, age, and occurrence of incidentcardiovascular events were available for 25,436. Of these, 24,320 wereCaucasian, 468 were African-American, 282 were Asian American, 262 whereHispanic, 62 were Native American, and 42 had unknown ancestry.

Statistical methods: Deviations from Hardy-Weinberg equilibrium wereevaluated using a log-likelihood ratio test. Differences in clinicalcovariates among the three genotype classes were assessed by ANOVAprocedures for normally-distributed characteristics, by theKruskal-Wallis test for non-normally distributed characteristics, and bya Chi-Squared test of proportions for categorical characteristics.Similarly, the significance of differences in allele frequency betweenpairs of populations with different biogeographic ancestry wasdetermined with a Chi-Squared test. For analysis of geographic bias inLp(a) levels and in the minor allele frequency of rs3798220 alleles,place of residence at enrollment was classified into one of six groupscorresponding to the northeast, south, midwest, southwest, mountain, andfar west areas of the United States. The significance of theseassignments in explaining Lp(a) levels or allele frequency wasdetermined with logistic regression or chi-square analysis. Prospectiveassessment of the association between rs3798220 and the risk of incidentCVD was performed with Cox proportional hazards models adjusted for ageor traditional risk covariates (age, blood pressure, history ofdiabetes, smoking status, familial history of myocardial infarction,LDL-C, and HDL-C). In all reported analysis, the assumption ofproportionality in the Cox models could not be rejected (p>0.05).Differences in risk associated with aspirin treatment were determinedeither by including an interaction term in the Cox models or by analysisthat stratified participants according to their assignment to aspirin orplacebo.

Examination of Lp(a) levels among Caucasian rs3798220 heterozygotesrevealed a clear bimodal distribution. One subgroup had lower Lp(a)levels with an approximately log-normal distribution that is alsoobserved for non-carriers of the minor allele, while the other subgrouphad higher Lp(a) levels with an approximate normal distribution.Therefore, these participants were classified into low or high Lp(a)subgroups by modeling the observed bimodal distribution as a mixture ofindependent log-normal [for low Lp(a)] and normal [for high Lp(a)]distributions that were fit to Lp(a) values with an expectationmaximization (EM) algorithm (10). An Lp(a) level of 27.9 mg/dLcorresponding to a responsibility value of 0.5 in the EM algorithm wasused to discriminate the high Lp(a) group from the low Lp(a) group. Theclassification with this two-distribution model was essentiallyunambiguous in that of 806 assignments out of 887 (91%) hadresponsibility values that were at least 95% of certainty.

REFERENCES FOR MATERIALS AND METHODS

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TABLE 1 Baseline clinical profile for Caucasian WHS participants bygenotype rs3798220 genotype* TT (N = 24119) TC (N = 904) CC (N = 15)p-value Clinical characteristics age (yrs.) 52 (48.0-59.0) 52.0(48-0-58.0) 52 (49.5-55.0) 0.352 BMI (kg/m{circumflex over ( )}2) 24.9(22.5-28.3) 24.7 (22.4-28.3) 24.9 (22.1-28.9) 0.758 history hypertension(%) 5950 (24.7) 237 (26.2) 3 (20.0) 0.530 family history MI (%) 2801(12.9) 127 (15.5) 2 (15.4) 0.077 current smoking (%) 2795 (11.6) 109(12.1) 2 (13.3) 0.912 diabetes (%) 614 (2.5) 22 (2.4) 0 (0.0) 0.944 HRT(%) 10472 (43.5) 419 (46.5) 9 (60.0) 0.100 menopause (%) 13139 (54.6)486 (53.8) 8 (53.3) 0.907 Lipid biomarkers total cholesterol (mg/dL)208.0 (184.0-235.0) 215.0 (189.0-241.0) 229 (208.5-250.0) 0.00031^(#)LDL-C (mg/dL) 121.3 (100.4-144.2) 126.5 (105.5-149.2) 137.6(118.8-157.4) 0.00022^(#) apolipoprotein B (mg/dL) 100.0 (83.8-121.3)107.3 (87.1-125.1) 109.8 (100.4-127.0) 0.00001^(#) HDL-C (mg/dL) 51.8(43.1-62.2) 51.8 (43.5-62.4) 51.7 (46.0-66.8) 0.600 apolipoprotein AI(mg/dL) 148.9 (132.3-167.9) 148.2 (133.4-166.3) 145.2 (135.9-168.3)0.983 triglycerides (mg/dL 119 (84-176) 117.0 (83.0-177.2) 104(90.0-149.0) 0.890 Lp(a) (mg/dL) 10.0 (4.2-28.5) 79.3 (13.7-102.1) 153.9(105.6-215.0) <<0.001⁺ Inflammation biomarkers C-reactive protein (mg/L)2.0 (0.8-4.4) 2.0 (0.8-4.2) 1.1 (0.9-2.4) 0.342 soluble ICAM-1 (ng/ml)342.9 (301.8-394.8) 343.8 (301.4-396.4) 349.4 (295.6-399.2) 0.961fibrinogen (mg/L) 349.9 (307.0-401.1) 348.9 (307.6-401.7) 343.5(318.4-382.7) 0.851 Other biomarkers creatinine (mg/dL) 0.7 (0.6-0.8)0.7 (0.6-0.8) 0.7 (0.7-0.8) 0.402 homocysteine (umol/dL) 10.5 (8.7-12.9)10.5 (8.7-12.8) 11.6 (8.6-12.7) 0.468 HbA1c (%) 5.0 (4.8-5.2) 5.0(5.8-5.2) 4.9 (4.8-5.2) 0.658 *Median (inter-quartile range forquantitative characteristics of N (%) for discrete characteristics.⁺Analytic p = 4.7 × 10⁻¹⁷⁷ ^(#)Not significant after adjustment forLp(a) levels.

TABLE 2 Association of rs3798220 with cardiovascular events amongCaucasians rs3798220 genotype and Lp(a) levels high Lp(a) low Lp(a) ntsHR CI) p HR CI) p HR CI) p Total incident CVD 825 1.502.05) 0.0121.782.51) 0.001 0.841.77) 0.650 Major incident CVD 521 1.582.33) 0.0211.852.83) 0.005 0.962.33) 0.930 Myocardial infarction 211 1.572.88)0.150 1.833.57) 0.076 0.953.83) 0.940 Ischemic stroke 221 1.813.17)0.039 2.013.79) 0.031 1.364.25) 0.600 Revascularization 475 1.432.18)0.092 1.792.80) 0.011 0.621.93) 0.410 Cox proportional hazards modelscomparing heterozygotes to the reference homozygotes for major allelegenotype (TT), including adjustments for age.

TABLE 3 Minor allele frequency of rs3798220 in WHS and HapMapsubpopulations p-value for pairwise difference in MAF group N⁺ MAF HWEpHisp. A. Am As N. Am. CHB + JPT CEU YRI WHS all 26495 0.021 0.002 — — —— — — — WHS Caucasian 25038 0.019 0.048 <0.001  0.001 <0.001 0.001<0.001 0.002 0.002 WHS Hispanic 275 0.151 0.901 — <0.001 <0.001 0.017 0.005 0.000 0.000 WHS African-American 486 0.005 0.872 — — <0.001 0.001<0.001 0.274 0.272 WHS Asian 360 0.076 0.940 — — — 0.244  0.593 0.0050.005 WHS Native American 66 0.091 0.273 — — — —  0.808 0.004 0.005HapMap CHB + JPT 89 0.056 0.440 — — — — — 0.056 0.055 HapMap CEU 600.000 — — — — — — — NA HapMap YRI 60 0.000 — — — — — — — — ⁺Number ofindividuals in each subpopulation with successful genotype for rs3798220NA, not applicable

Example 2: Genotyping of 40 Additional SNPs at and Near the LPA Locus inWHS Samples to Determine SNP Variation in LD with Rs3798220 inEuropean-Americans, Asian-Americans, and American Hispanics

We genotyped 40 additional SNPs at LPA (the gene for apolipoprotein (a))and neighboring loci in a subset of samples from the WHS chosen on thebasis of both rs3798220 genotype and self-reported ancestry(European-American (i.e., White or Caucasian), Asian-American, orAmerican Hispanic). We then estimated linkage disequilibrium (LD)between these new SNPs and rs378220. Because the samples were not chosenat random but instead on the basis of rs3798220 genotype, we could notsimply estimate allele frequency and LD from the sample directly but hadto infer allele frequencies and LD by minimizing the difference betweenthe observed and predicted genotypes given the rs379220-based samplingstrategies. The results showed that:

1) two SNPs (rs9457931 dbSNP@NCBI (single nucleotide polymorphism atposition chromosome 6:160849894 NCBI build 128) and rs9457927 dbSNP@NCBI(single nucleotide polymorphism at position chromosome 6:160830272 NCBIbuild 128)) were in almost complete LD with rs3798220 among the WHSCaucasians (European-Americans). Both SNPs are in neighboring LPAL2 gene(lipoprotein, Lp(a)-like 2 precursor gene, adjacent to the gene forapolipoprotein(a));

2) other SNPs have a range of LD to rs3798220; and

3) there is a very different pattern of LD in the non-Europeansub-populations. We interpret these results to suggest that the effectswe described in Example 1 are related to an allele carrying all threeSNPs (rs3798220, rs9457931, rs9457927).

Example 3: Relationship Between Rs3798220 Genotype, Number of Kringle IVType 2 Repeats and Lp(a) Expression in Three Sub-Groups from the DallasHeart Study with Distinct Ancestry

We had rs3798220 genotyped in Dallas Heart Study (DHS) (The AmericanJournal of Cardiology Volume 93, Issue 12, 15 Jun. 2004, Pages1473-1480) for which the number of Kr IV, type 2 repeats (KrIV2r's) inthe apolipoprotein(a) component of plasma Lp(a) (i.e. expressed apop(a)protein) had been determined previously (Circulation. 2005;111:1471-1479). This population includes both men and women, amongnon-Hispanic Whites (European ancestry), Blacks (African ancestry), andHispanics, totaling 3529 samples. The analysis sought to identify acorrespondence between the minor allele of rs3798220 and the number ofexpressed KrIV2r's, with possible dependence on ancestry. The analysiswas performed the analysis two ways. In the first, all individuals wereconsidered, and each individual's KrIV2r alleles (at the DNA level) wereassumed to be equivalent to the expressed alleles (at the proteinlevel). That is, individuals with only one KrIV2r expressed were assumedto have two copies of that allele at the LPA locus (3529 individuals).In the second, we considered only individuals with two clearly distinctKrIV2r alleles (2859 total), excluding 668 with only one KrIV2r alleleexpressed from analysis. The conclusions were very similar for both.Namely, 1) among non-Hispanic whites, the minor allele of rs3798220 wasstrongly correlated with 17 KrIV2r repeats and to a lesser extent 16 or18 repeats (although the difference between 16, 17 and 18 repeats couldrelate to the resolution of the assay for KrIV2r number) (FIGS. 3A-3B);2) among non-Hispanic whites, alleles with a smaller number of KrIV2r'sgenerally express higher levels of Lp(a) (FIG. 4 ); 3) whilenon-Hispanic whites expressing 17 KrIV2r's have high levels of Lp(a),individuals who, in addition, also carry one copy of the minor allele ofrs3798220 have significantly even higher levels of Lp(a) (page 11, lowerright) (FIG. 5 ); and 4) the distribution of KrIV2r's, their correlationwith expression level, and their correlation with rs3798220 is differentin the different ancestral groups so that correlation of rs3798220 andextreme levels of Lp(a) observed non-Hispanic whites is not observed inthe groups of the other ancestries.

We claim:
 1. A method comprising: (a) determining the identity of asingle nucleotide polymorphism at position chromosome 6:160880877 (March2006 assembly—NCBI build 36.1; rs3798220 dbSNP @ NCBI) of anapolipoprotein(a) (Apo(a)) gene in a human subject, wherein the presenceof cytosine or guanine at position chromosome 6:160880877 indicatesresponsiveness to acetylsalicylic acid, wherein the presence of thymineor adenine at position chromosome 6:160880877 indicatesnon-responsiveness to acetylsalicylic acid; and (b) administeringacetylsalicylic acid to a human subject responsive to acetylsalicylicacid to reduce the risk of a cardiovascular event, or administering anantiplatelet or antithrombotic agent that is not acetylsalicylic acid toa human subject non-responsive to acetylsalicylic acid to reduce therisk of a cardiovascular event.
 2. The method of claim 1, furthercomprising determining a level of Lipoprotein(a) (Lp(a)) in a bloodsample from the subject.
 3. The method of claim 2, wherein the subjecthas an elevated level of Lp(a) in the blood.
 4. The method of claim 3,wherein the level of Lp(a) is about 10 mg/dl or higher in the bloodsample from the subject.
 5. The method of claim 3, wherein the level ofLp(a) is about 15 mg/dl or higher in the blood sample from the subject.6. The method of claim 3, wherein the level of Lp(a) is about 20 mg/dlor higher in the blood sample from the subject.
 7. The method of claim3, wherein the level of Lp(a) is about 25 mg/dl or higher in the bloodsample from the subject.
 8. The method of claim 1, wherein thecardiovascular event is myocardial infarction, stroke, acute coronarysyndrome, myocardial ischemia, chronic stable angina pectoris, unstableangina pectoris, cardiovascular death, coronary re-stenosis, coronarystent re-stenosis, coronary stent re-thrombosis, revascularization,angioplasty, transient ischemic attack, pulmonary embolism, vascularocclusion, or venous thrombosis.
 9. The method of claim 1, wherein theidentity of the polymorphism is determined by contacting a nucleic acidobtained from the subject with a nucleic acid probe.
 10. The method ofclaim 1, wherein the identity of the polymorphism is determined byallele-specific probe hybridization, allele-specific primer extension,allele-specific amplification, 5′ nuclease digestion, molecular beaconassay, oligonucleotide ligation assay, size analysis, or single-strandedconformation polymorphism.
 11. The method of claim 1, wherein theidentity of the polymorphism is determined by sequencing a nucleic acidobtained from the subject.
 12. A method of treatment comprising:selecting a human subject on the basis that the human subject has anApo(a) polymorphism characterized by cytosine or guanine at chromosome6:160880877 (March 2006 assembly—NCBI build 36.1; rs3798220 dbSNP @NCBI), and administering to the human subject acetylsalicylic acid,thereby reducing the risk of a future cardiovascular event.
 13. Themethod of claim 12, wherein the human subject also has an elevated levelof Lipoprotein(a) (Lp(a)) in the blood.
 14. The method of claim 13,wherein the level of Lp(a) is about 10 mg/dl or higher in a blood samplefrom the subject.
 15. The method of claim 14, wherein the level of Lp(a)is about 15 mg/dl or higher in a blood sample from the subject.
 16. Themethod of claim 12, wherein the cardiovascular event is myocardialinfarction, stroke, acute coronary syndrome, myocardial ischemia,chronic stable angina pectoris, unstable angina pectoris, cardiovasculardeath, coronary re-stenosis, coronary stent re-stenosis, coronary stentre-thrombosis, revascularization, angioplasty, transient ischemicattack, pulmonary embolism, vascular occlusion, or venous thrombosis.